摘要:

AbstractWe have developed an in vitro model incorporating a low‐amplitude (10−7V/cm), low frequency (f<100 Hz), capacitively coupled electric field in order to study the mechanism through which an electric field may increase bone cell proliferation. Utilizing this model we have previously shown that electric field‐stimulated bone cell proliferation was dependent on release of mitogen activity into the culture medium from exposed cells. The current studies were intended to characterize this mitogen activity. In these studies we found that electric field‐stimulated human bone cell proliferation was associated with increased IGF‐II mRNA accumulation and IGF‐II secretion suggesting that IGF‐II may in part mediate the increase in bone cell proliferation following electric f

ISSN:0021-9541    

DOI:10.1002/jcp.1041500112

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe aim of the present study was to investigate the relationship between agonist‐induced changes in intracellular free Ca2+([Ca2+]j) and the refilling of intracellular Ca2+stores in Fura 2–loaded thyroid FRTL‐5 cells. Stimulating the cells with ATP induced a dose‐dependent increase in ([Ca2+]j). The ATP‐induced increase in [Ca2+]jwas dependent on both release of sequestered intracellular Ca2+as well as influx of extracellular Ca2+. Addition of Ni2+prior to ATP blunted the component of the ATP‐induced increase in [Ca2+]jdependent on influx of Ca2+. In cells stimulated with ATP in a Ca2+‐free buffer, readdition of Ca2+induced a rapid increase in [Ca2+]j; this increase was inhibited by Ni2+. In addition, the ATP‐induced influx of45Ca2+was blocked by Ni2+. Stimulating the cells with noradrenaline (NA) also induced release of sequestered Ca2+and an influx of extracellular Ca2+. When cells were stimulated first with NA, a subsequent addition of ATP induced a blunted increase in [Ca2+]j. If the action of NA was terminated by addition of prazosin, and ATP was then added, the increase in [Ca2+]jwas restored to control levels. Addition of Ni2+prior to prazosin inhibited the restoration of the ATP response. In the presence of extracellular Mn2+, ATP stimulated quenching of Fura 2 fluorescence. The quenching was probably due to influx of Mn2+, as it was blocked by Ni2+. The results thus suggested that stimulating release of sequestered Ca2+in FRTL‐5 cells was followed by influx of extracellular Ca2+and rapid refilling of intrace

ISSN:0021-9541    

DOI:10.1002/jcp.1041500113

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe progression of T‐lymphoma cells (CCRF‐CEM) growing in suspension has been monitored during long term (12–28 h) batch experiments using microcalorimetry. In parallel with the calorimetric measurements, changes in cell concentration, pH,p(O2) and concentrations of the main energy sources (glucose and glutamine) were determined. The overall metabolic rate per cell (as reflected by the heat production rate per cell,Pcell) and the growth rate decreased with time. These changes could be attributed solely to the decrease in pH of the medium until the total heat production,Q, exceeded 1.2 J per ml (corresponding to an incubation time of 20 h of a batch having an initial cell concentration of 1 x 106cells per ml). The lowering ofp(O2) to a level of 0.02 mmol/l or the decrease in concentrations of glucose and glutamine to7.7and 1.3 mmol/l, respectively, did not influencePcellor the growth pattern. No “crowding effect” was observed for the cells in the investigated concentration range (0.6–1.3) × 106

ISSN:0021-9541    

DOI:10.1002/jcp.1041500114

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractA decline in cell surface γ‐glutamyl transpeptidase specific activity was previously observed to be concomitant with C6glial cell proliferation. To elucidate the underlying factor(s) mediating γ‐glutamyl transpeptidase down‐regulation, the effects of C6cell density and culture conditions on cell surface transpeptidase activity levels were investigated. After 24 h of culture, the transpeptidase specific activities were inversely related to the initial plating densities. The lower‐density cultures showed an induction within 24 h of plating. As the cultures proliferated, the specific transpeptidase activities declined to a common low level at postconfluency. The γ‐glutamyl transpeptidase down‐regulation was unrelated to cell growth rate and was most pronounced during logarithmic proliferation. Induction and down‐regulation of γ‐glutamyl transpeptidase activity at low cell densities were not a result of trypsinization. Supplementation of low‐density cultures with conditioned medium, use of matrix‐coated wells, or periodic replacement of growth media to prevent conditioning had minor effects on the decline of cell surface activity. Kinetic analysis showed that the Michaelis constants and the reaction mechanism were unaltered by cell density, indicating that downregulation was not due to allosteric factors or an alteration in enzyme character. A reduction in the maximal velocity of cell surface transpeptidation at higher cell densities suggested that γ‐glutamyl transpeptidase down‐regulation is related to the concentration of enzyme at the cell surface. Immunocytochemical localization of γ‐glutamyl transpeptidase demonstrated that γ‐glutamyl transpeptidase antigen levels decrease as C6cell density increases. These results led us to propose that cell‐cell contact stimulates the disappearance of γ‐glutamyl transpeptidase fro

ISSN:0021-9541    

DOI:10.1002/jcp.1041500115

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractHepatocyte growth factor (HGF) also known as hepatopoietin A (HPTA) (Michalopoulos, FASEB J., 4:176–187, 1990) is a heparin‐binding growth factor whose characterization and tissue distribution have been reported elsewhere. This growth factor was recently cloned and its amino acid sequence determined under the name of hepatocyte growth factor (HGF) (Miyazawa et al., Biochem. Biophys. Res. Commun.,163:967–973, 1989; Zarnegar et al., Biochem. Biophys. Res. Commun.,163:1370–1376, 1989; Nakamura et al., Nature,342:440–443, 1989). Human placenta is one of the tissues that contains significant amounts of HGF. We isolated HGF from human placenta and characterized its biologic effect on rat hepatocytes. Human placenta HGF was isolated in high purity as a single chain molecule. Single chain HGF stimulated DNA synthesis in primary rat hepatocyte cultures in serum‐free medium. The maximal effect was seen at 5–10 ng/ml. The maximal response occurred at 25–48 hours after plating of the hepatocytes. Protein synthesis was also stimulated by HGF in primary rat hepatocyte cultures. There were peak responses at 19–24 and 37–42 hours after plating of the hepatocytes. TGFβ1inhibited more than 95% of HGF‐induced DNA synthesis but only 25% of HGF‐induced protein synthesis. HGF interacted in an additive manner with EGF, a well‐known hepatocyte mitogen. There was not an additive interaction between HGF and aFGF. Regenerating liver hepatocytes obtained from rats which underwent two‐thirds partial hepatectomies (PHX) also responded to HGF in a dose‐dependent manner as the

ISSN:0021-9541    

DOI:10.1002/jcp.1041500116

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWe have studied the effect of intracellular pH on the establishment and maintenance of the cellular polarity in MDCK cells by utilizing nigericin which causes lowering of the cytoplasmic pH. At pH below 6.5, MDCK cells lost their polarized morphology and became roundish, with an increased apical area and shortened and unstable lateral walls. The lateral wall marker proteins uvomorulin and Na, K‐ATPase remained segregated to the lateral walls in the acidified cells, as shown by immunofluorescence microscopy. Fodrin, on the other hand, was released from its normal basolateral residence and was found in the cytoplasm. Actin, which normally co‐localizes with fodrin along the basolateral walls, showed a dotty distribution in the cytoplasm of acidified cells, while stress fibers remained intact. Microtubular network appeared flattened, but the Golgi complex retained its apical position. The pH change–induced alterations were readily reversible, as the normal basal‐apical polarity (columnar shape, distinct apical and lateral domains with apposing and stiff lateral membranes) was reformed within 10 minutes after restoring the normal pH gradient across the cell membrane. This coincided with the translocation of fodrin from the cytoplasm to the lateral walls. The results show that lowering of intracellular pH leads to temporary segregation of fodrin from the other components of the membrane skeleton assembly, and that association of fodrin with the lateral walls seems to be a prerequisite for their close apposition and for the maintenance of normal basal‐axial

ISSN:0021-9541    

DOI:10.1002/jcp.1041500117

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractIn a previous study, we revealed that tumor necrosis factor (TNF) was secreted in mouse liver at an early phase of liver regeneration after partial hepatectomy. Here, we investigated direct actions of TNF on the in vitro DNA synthesis of adult mouse hepatocytes in primary culture. TNF enhanced both3H‐TdR uptake and the number of3H‐TdR‐labeled nuclei of hepatocytes. Their time courses were similar to those by epidermal growth factor (EGF) with about a 15 h lag period and a peak period of 24–48 h. This action of TNF was abrogated by DNA polymerase α inhibitor, aphidicolin and blocked specifically by anti‐TNF antibody. The actions of rm TNF were not distinguishable; ED50 was about 7.5U/ml (5ng/ml) and 30U/ml (20ng/ml) for maximal response (about 2‐fold or more of control). Other inflammatory monokines showed differential effects on in vitro DNA synthesis of hepatocyte. Neither type of interleukin 1 affected hepatocyte DNA synthesis in the range examined (up to 50 ng/ml). IL‐6 markedly inhibited the hepatocyte DNA synthesis stimulated by TNF and EGF. The action of TNF was completely suppressed by transforming growth factor β, which is known as a potent inhibitor of hepatocyte growth. Interferon γ also blocked this TNF action when added simultaneously. These results indicate that the activation of tissue macrophages and local secretion of TNF in liver after partial hepatectomy is of physiological importance in liver regeneration, in part by a direct stimulation of hepatocyte DNA synthesis. Cytokines induced by TNF may also participate in the later termination of li

ISSN:0021-9541    

DOI:10.1002/jcp.1041500118

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractCyclic nucleotide phosphodiesterase activity of human peripheral blood mononuclear cells was significantly increased following a short (30 min) incubation with the mitogenic lectin Concanavalin A. Con A stimulated phosphodiesterase activity to the same extent whatever the subcellular compartment (homogenate, cytosol or pellet). Further separation of the Con A‐activated mononuclear cells into lymphocyte‐enriched and monocyte‐enriched populations showed that the Con A‐induced increase of phosphodiesterase activity exclusively affected the lymphocyte‐enriched population. In lymphocytes, cyclic GMP phosphodiesterase activity was more importantly enhanced by Con A ( + 275%) than cyclic AMP phosphodiesterase activity (+ 75%). The increase of both activities occurred as early as from 10 min of Con A incubation and proved to be maximal with Con A doses of 2.5 and 5 μg per 106cells, lower and higher doses being less effective. Inhibition experiments with reference inhibitors suggested that, among the high affinity phosphodiesterase isoforms, the cyclic GMP‐inhibited enzyme might be more selectively enhanced by Con A than the cyclic AMP‐specific, Rolipramsensitive one. The non‐mitogenic lectin Helix pomatia hemagglutinin, was not able to enhance cyclic nucleotide phosphodiesterase activity of human mononuclear cells whereas anti‐CD3 monoclonal antibody, although being less effective than Con A, exhibited a significant stimulatory effect. Putting together these results suggest that the early increase in phosphodiesterase activity might be a normal step involved in the mitogenic activation o

ISSN:0021-9541    

DOI:10.1002/jcp.1041500119

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe role of amino acids in the regulation of RNA degradation was investigated in cultured hepatocytes from fed rats previously labeled in vivo with [6‐14C]orotic acid. Rates of RNA degradation were determined between 42 and 48 h of culture from the release of radioactive cytidine in the presence of 0.5 mM unlabeled cytidine. The fractional rate was about 4.4 ± 0.4%/h in the absence of amino acids (0×). The catabolism of RNA was decreased to basal level (1.5 ± 0.3%/h) by the addition of amino acids at 10 times normal plasma concentration (10 x). The inhibition of RNA degradation, expressed as percentage of maximal deprivation‐induced response (0× minus 10×), averaged 60% at normal plasma levels of amino acids. The degree of responsiveness was greatly improved as compared to freshly isolated hepatocytes (20%) and was similar to the sensitivity previously observed with perfused livers. In cultured hepatocytes, the sensitivity of RNA degradation to amino acids was not affected by varying the volume of medium from 1 to 4 ml per dish. In freshly isolated hepatocytes, the inhibitory effect of amino acids was not modified by changing the cell density from 0.5 to 5 × 106cells per ml. In the range of normal plasma concentration of amino acids, the low sensitivity of RNA degradation in isolated hepatocytes persisted with inhibition ranging from 10 to 20%. These findings suggest that the control of RNA degradation in both cultured and isolated hepatocytes is not affected by the total quantity of amino acids available in the medium, but their concentration is crucial. Electron microscopy observations and the inhibitory effect of 3‐methyladenine in cultured rat hepatocytes partially confirmed the role of the lysosomal system in the increase of RNA degradation and its regulation by

ISSN:0021-9541    

DOI:10.1002/jcp.1041500120

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractCell lines were established from rabbit articular chondrocytes following transfection with a plasmid encoding SV40 early function genes. This resulted in cell immortalization (130 passages have been completed for the oldest cell line) with acquisition of characteristics of partial transformation such as reduced serum requirements for normal and clonal growth. The immortalized chondrocytes, called SVRAC, did not form multilayer foci when maintained in postconfluent culture. Their ability to form colonies in soft agar was not increased in comparison with normal chondrocytes, but they were weakly tumorigenic in nude mice. SVRAC lost the ability to synthesize type II collagen and Alcian blue–stainable matrix, which are markers of the differentiated chondrocyte phenotype, and synthesized predominantly type I collagen. Studies of collagen gene expression showed that proα1 (II) mRNA was undetectable, whereas proα1 (I) collagen mRNA was expressed even in late passage cultures. Unlike normal dedifferentiated chondrocytes, SVRAC were unable to re‐express the differentiated phenotype in response to tridimensional culture or microfilament depolymerization. Cell lines obtained from chondrocytes transfected either in primary culture or just after release of cells from cartilage displayed the same behaviour. Thus SV40 early genes were able to immortalize rabbit articular chondrocytes, but the resulting cell lines displayed an apparently irreversibly dedifferentiated phenotype. These cell lines can be used as models to identify regulatory pathways that are required for the maintenance or reexpression of differentiated function in chondro

ISSN:0021-9541    

DOI:10.1002/jcp.1041500121

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY